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Thy-1 Is Expressed in Hepatic Myofibroblasts and Not Oval Cells in Stem Cell-Mediated Liver Regeneration

2007; Elsevier BV; Volume: 171; Issue: 5 Linguagem: Inglês

10.2353/ajpath.2007.070273

1525-2191

Katalin Dezső, Peter Jelnes, Viktória László, Kornélia Baghy, Csaba Bödör, Sándor Paku, Niels Tygstrup, Hanne Cathrine Bisgaard, Péter Nagy,

Organ Transplantation Techniques and Outcomes

Thy-1, a marker of hematopoietic stem cells, has been reported to be expressed by oval cells proliferating during stem cell-mediated regeneration in rat liver, suggesting a relationship between the two cell populations. Consequently, Thy-1 has become an accepted cell surface marker to sort hepatic oval cells. In the present study we used the well-characterized 2-acetylaminfluorene/partial hepatectomy model to induce transit-amplification of hepatic oval cells in the regenerating liver and characterized Thy-1 expression using Northern hybridization, quantitative reverse transcriptase-polymerase chain reaction analysis, immunofluorescence confocal microscopy, and immunoelectronmicroscopy. We found that Thy-1 expression was induced during transit-amplification of the oval cell population, but Thy-1 mRNA was not present in the α-fetoprotein-expressing oval cells. Thy-1 protein was consistently present outside the basement membrane surrounding the oval cells. It overlapped frequently with smooth muscle actin staining. A similar cellular localization of the Thy-1 protein was found on human liver specimens with ductular reactions obtained from patients with fulminant liver failure. Furthermore, Thy-1 was expressed by myofibroblasts in experimental liver fibrosis models without oval cell proliferation. We conclude that Thy-1 is not a marker of oval cells but is present on a subpopulation of myofibroblasts/stellate cells. Thy-1, a marker of hematopoietic stem cells, has been reported to be expressed by oval cells proliferating during stem cell-mediated regeneration in rat liver, suggesting a relationship between the two cell populations. Consequently, Thy-1 has become an accepted cell surface marker to sort hepatic oval cells. In the present study we used the well-characterized 2-acetylaminfluorene/partial hepatectomy model to induce transit-amplification of hepatic oval cells in the regenerating liver and characterized Thy-1 expression using Northern hybridization, quantitative reverse transcriptase-polymerase chain reaction analysis, immunofluorescence confocal microscopy, and immunoelectronmicroscopy. We found that Thy-1 expression was induced during transit-amplification of the oval cell population, but Thy-1 mRNA was not present in the α-fetoprotein-expressing oval cells. Thy-1 protein was consistently present outside the basement membrane surrounding the oval cells. It overlapped frequently with smooth muscle actin staining. A similar cellular localization of the Thy-1 protein was found on human liver specimens with ductular reactions obtained from patients with fulminant liver failure. Furthermore, Thy-1 was expressed by myofibroblasts in experimental liver fibrosis models without oval cell proliferation. We conclude that Thy-1 is not a marker of oval cells but is present on a subpopulation of myofibroblasts/stellate cells. Thy-1 (CD-90) is a rather promiscuous molecule. It is expressed by several different cell types, and, among others, it is present on the surface of the bone marrow stem cells. It was also reported to be present in the rat liver on the oval/progenitor cells in stem cell-mediated liver regeneration.1Petersen BE Goff JP Greenberger JS Michalopoulos GK Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat.Hepatology. 1998; 27: 433-445Crossref PubMed Scopus (337) Google Scholar, 2Shu SN Wei L Wang JH Zhan YT Chen HS Wang Y Hepatic differentiation capability of rat bone marrow-derived mesenchymal stem cells and hematopoietic stem cells.World J Gastroenterol. 2004; 10: 2818-2822Crossref PubMed Scopus (83) Google Scholar, 3Laurson J Selden C Hodgson HJ Hepatocyte progenitors in man and in rodents—multiple pathways, multiple candidates.Int J Exp Pathol. 2005; 86: 1-18Crossref PubMed Scopus (40) Google Scholar, 4Pi L Oh SH Shupe T Petersen BE Role of connective tissue growth factor in oval cell response during liver regeneration after 2-AAF/PHx in rats.Gastroenterology. 2005; 128: 2077-2088Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar Later, a precursor-product relationship was described between bone marrow cells and oval cells/hepatocytes in several experimental models1Petersen BE Goff JP Greenberger JS Michalopoulos GK Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat.Hepatology. 1998; 27: 433-445Crossref PubMed Scopus (337) Google Scholar, 3Laurson J Selden C Hodgson HJ Hepatocyte progenitors in man and in rodents—multiple pathways, multiple candidates.Int J Exp Pathol. 2005; 86: 1-18Crossref PubMed Scopus (40) Google Scholar, 5Theise ND Krause DS Bone marrow to liver: the blood of Prometheus.Semin Cell Dev Biol. 2002; 13: 411-417Crossref PubMed Scopus (40) Google Scholar, 6Petersen BE Bowen WC Patrene KD Mars WM Sullivan AK Murase N Boggs SS Greenberger JS Goff JP Bone marrow as a potential source of hepatic oval cells.Science. 1999; 284: 1168-1170Crossref PubMed Scopus (2205) Google Scholar as well as in humans,7Masson NM Currie IS Terrace JD Garden OJ Parks RW Ross JA Hepatic progenitor cells in human fetal liver express the oval cell marker Thy-1.Am J Physiol Gastrointest Liver Physiol. 2006; 291: G45-G54Crossref PubMed Scopus (81) Google Scholar raising the very exiting possibility of liver cells being derived from hematopoietic cells. Several groups confirmed the Thy-1 expression in oval cells,1Petersen BE Goff JP Greenberger JS Michalopoulos GK Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat.Hepatology. 1998; 27: 433-445Crossref PubMed Scopus (337) Google Scholar, 2Shu SN Wei L Wang JH Zhan YT Chen HS Wang Y Hepatic differentiation capability of rat bone marrow-derived mesenchymal stem cells and hematopoietic stem cells.World J Gastroenterol. 2004; 10: 2818-2822Crossref PubMed Scopus (83) Google Scholar, 3Laurson J Selden C Hodgson HJ Hepatocyte progenitors in man and in rodents—multiple pathways, multiple candidates.Int J Exp Pathol. 2005; 86: 1-18Crossref PubMed Scopus (40) Google Scholar, 4Pi L Oh SH Shupe T Petersen BE Role of connective tissue growth factor in oval cell response during liver regeneration after 2-AAF/PHx in rats.Gastroenterology. 2005; 128: 2077-2088Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar resulting in the extensive use of Thy-1 as a cell surface marker to sort out liver progenitor cells. However, the issue of stem cell transdifferentiation has subsequently been one of the most debated issues in hepatic pathobiology, and most of these observations can now be explained by cell fusion and not transdifferentiation. The most comprehensive review of this topic recently concluded that although “data are sufficient to indicate that mesodermal hematopoietic cells can generate hepatocytes at a very low frequency, this is not an effective pathway under most conditions.”8Thorgeirsson SS Grisham JW Hemopoietic cells as hepatocyte stem cells: a critical review of the evidence.Hepatology. 2006; 43: 2-8Crossref PubMed Scopus (206) Google Scholar At the same time, others described cells coexpressing Thy-1 and smooth muscle actin (SMA) in similar experimental settings,9Hoppo T Fujii H Hirose T Yasuchika K Azuma H Baba S Naito M Machimoto T Ikai I Thy1-positive mesenchymal cells promote the maturation of CD49f-positive hepatic progenitor cells in the mouse fetal liver.Hepatology. 2004; 39: 1362-1370Crossref PubMed Scopus (64) Google Scholar questioning the identity of the Thy-1-expressing cells in the liver. To resolve this contradiction we performed detailed morphological expression analysis to identify the location of Thy-1 in the normal liver and in damaged liver with and without oval cell proliferation. Male F-344 rats (160 to 180 g) were used for all experiments and were kept under standard conditions. Animal protocols were approved by the Danish Council for Supervision with Experimental Animals. The animals received 2-acetylaminofluorene (AAF) (suspended in 1% dimethylcellulose) at 4.5, 9, 12, or 18 mg/kg/day administered daily for 4 consecutive days by gavage. Traditional two-thirds partial hepatectomy (PHx)10Higgins GM Anderson RM Experimental pathology of the liver: restoration of liver of white rat following partial surgical removal.Exp Pathol. 1931; 12: 186-202Google Scholar was performed on the 5th day, followed by four additional AAF treatments. Groups of three animals were sacrificed 1, 5, 9, 14, and 21 days after PHx. Controls included untreated animals and rats subjected to a PHx or a sham laparotomy only. After resection of the liver, samples were taken for histological examinations and the rest snap-frozen in liquid nitrogen for RNA extraction. Ligation of the common bile duct was done according to Cameron and Oakley.11Cameron GR Oakley CR Ligation of the common bile duct.J Pathol Bacteriol. 1932; 35: 769-798Crossref Google Scholar The rats were sacrificed 2 weeks after the operation. Twenty percent CCl4 (0.5 ml/kg, dissolved in vegetable oil) was administered by gavage to rats twice a week while the animals were kept on 0.05% phenobarbital in the drinking water. The experiment was terminated after 8 weeks.12Proctor E Chatamra K High yield micronodular cirrhosis in the rat.Gastroenterology. 1982; 83: 1183-1190Abstract Full Text PDF PubMed Scopus (268) Google Scholar Snap-frozen human liver specimens for immunohistochemical examination were obtained from two patients who underwent orthotopic liver transplantation because of fulminant liver failure of unknown etiology. The procedure was approved by the ethical committee of the Semmelweis University. Isolation of oval cells was performed using control liver, and animals were treated according to the AAF/PHx protocol (18 mg/kg/day) and sacrificed at day 9 after PHx. The isolation and enrichment procedure has been described in detail.13Bisgaard HC Santoni-Rugiu E Nagy P Thorgeirsson SS Modulation of the plasminogen activator/plasmin system in rat liver regenerating by recruitment of oval cells.Lab Invest. 1998; 78: 237-246PubMed Google Scholar In brief, liver cells were released by a three-step perfusion procedure in situ. Viable nonparenchymal cell populations were purified by centrifugation through a two-step Percoll gradient. Kupffer cells were removed by selective adherence to plastic tissue culture dishes. Removal of macrophages, endothelial cells, and red blood cells was achieved by selective panning using the mouse monoclonal antibody OX43 (catalog no. MCA276; Serotec, Oxford, UK). Cell preparations were snap-frozen in liquid nitrogen and stored at −70°C until processed for total RNA isolation and Northern blot analysis. Northern blotting with cDNA probes was performed as previously described.14Bisgaard HC Müller S Nagy P Rasmussen LJ Thorgeirsson SS Modulation of the gene network connected to interferon-γ in liver regeneration from oval cells.Am J Pathol. 1999; 155: 1075-1085Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar The cDNA for rat Thy-1 encompassed nucleotides 46 to 531 (GenBank accession no. NM_012673), and for α-fetoprotein (AFP), nucleotides 101 to 329 (GenBank accession no. X02361). The filters were hybridized with rat S18 to assess the integrity and ensure equal loading of the RNA. Frozen sections (8 μm) were fixed in acetone, dried at room temperature, and stained with RNase-free hematoxylin. Laser microdissection of oval cells was performed by using the PALM MicroBeam system, and 500 to 1000 cells were collected in RNA-Later. For whole liver quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis, frozen sections from normal and AAF/PHx-treated liver were collected in lysis buffer. Total RNA was isolated by RNAqueous micro kit (catalog no. AM 1931; Ambion, Austin, TX). A high capacity cDNA reverse transcription kit (catalog no. 4368814; ABI) was used for cDNA synthesis as recommended by the supplier. PCR was performed by the ABI Prism 7300 sequence detection system (Applied Biosystems, Weiterstadt, Germany), using ABI TaqMan gene expression assays for AFP (assay ID: Rn00560661_m1), SMA (assay ID: Rn01759928_ g1), and Thy-1 (assay ID: Rn00562048_m1) according to the manufacturer's instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control. All samples were run in triplicate, in a 20-μl reaction volume. Results were obtained as threshold cycle (CT) values. Expression levels were calculated using the ΔCT method. The values were calculated as the mean values of three independent measurements, and the expression levels of mRNA in all samples were defined as a ratio to GAPDH expression. Frozen sections (10 to 20 μm) were fixed in methanol and were incubated at room temperature (1 hour) with a mixture of the primary antibodies (Table 1) and with appropriate secondary antibodies afterward (Jackson ImmunoResearch, West Grove, PA). All samples were analyzed by confocal laser-scanning microscopy using the Bio-Rad MRC-1024 system (Bio-Rad, Richmond, CA). Negative controls were performed by replacing the primary antibodies with preimmune sera (data not shown).Table 1Primary Antibodies Used for the Immunohistochemical StudiesAntibodySpeciesManufacturer*Catalog numberDilutionLamininRabbit polyclonalDAKOZ00971:200Anti-rat Thy-1Mouse monoclonalBD Pharmingen5548951:100FITC-labeled anti-rat Thy-1Mouse monoclonalBD Pharmingen5548971:50Anti-human Thy-1Mouse monoclonalBD Pharmingen5504021:100FITC-labeled anti-human Thy-1Mouse monoclonalBD Pharmingen5555951:50GFAPMouse monoclonalBD Pharmingen5563301:100Anti-human cytokeratin-19Mouse monoclonalBioGenexMU246-UC1:50Anti-rat cytokeratin-19Mouse monoclonalNovocastraNCL-CK191:50OV-6Mouse monoclonalR&D SystemsMAB20201:100FITC-labeled cytokeratinMouse monoclonalDAKOF08591:10DesminRabbit polyclonalNeomarkersRB-9014-P11:100Smooth muscle actinMouse monoclonalDAKOM08511:100OX-62Mouse monoclonalSerotecMCA1029G1:100Mononuclear phagocyte (rMPh/ED-1)Mouse monoclonalBD Pharmingen5549541:100Lyve-1Rabbit polyclonalReliatech102-PA5051:100CD45Mouse monoclonalBD Pharmingen5505661:100DAKO, Glostrup, Denmark; BD Pharmingen, San Jose, CA; Biogenex, San Ramon, CA; Novocastra, Newcastle upon Tyne, UK; R & D System, Minneapolis, MN; Neomarkers, Fremont, CA; Serotec, Oxford, UK; Reliatech, Bvaunschweig, Germany. Open table in a new tab DAKO, Glostrup, Denmark; BD Pharmingen, San Jose, CA; Biogenex, San Ramon, CA; Novocastra, Newcastle upon Tyne, UK; R & D System, Minneapolis, MN; Neomarkers, Fremont, CA; Serotec, Oxford, UK; Reliatech, Bvaunschweig, Germany. Co-localization analysis was performed using the Image J program (National Institutes of Health, Bethesda, MD). The red (channel 1) and green (channel 2) images were acquired separately and sequentially to avoid bleed-through. The area fraction (%) occupied by red and green fields was determined by manual thresholding. Analysis of co-localized points (%) was determined using the co-localization plug-in. Preparation of liver tissue for immunoelectronmicroscopy was described by Paku and colleagues.15Paku S Schnur J Nagy P Thorgeirsson SS Origin and structural evolution of the early proliferating oval cells in rat liver.Am J Pathol. 2001; 158: 1313-1323Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar Cryosections were rinsed in phosphate-buffered saline and incubated with the primary antibody Thy-1 (dilution 1:100, 3 hours), followed by peroxidase-conjugated anti-mouse antibody (dilution 1:500, catalog no. 715-035-1500; Jackson ImmunoResearch). Semithin sections were slightly stained by 0.5% toluidine blue (pH 8.5), and unstained ultrathin sections were analyzed on a Philips CM 10 electron microscope (Philips, Eindhoven, The Netherlands). Transcripts for Thy-1 were not detected by Northern blot analysis in mRNA preparations from whole normal liver (Figure 1A) and were undetectable in preparations of nonparenchymal cells isolated from normal liver and enriched with a protocol for oval cells (Figure 1B). Likewise, qRT-PCR analysis detected low AFP, Thy-1, and SMA expression in normal liver (Figure 2).Figure 2qRT-PCR analysis of AFP, Thy-1, and SMA mRNA in whole liver of normal and AAF/PHx (9 days after PHx)-treated animals and in microdissected oval cell populations. The relative expression levels of AFP, Thy-1, and SMA were determined by comparing with that of GAPDH expression level (100%).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Thy-1 expression by immunohistochemistry was detectable and confined to the periportal region (Figure 3, A, B, C, and E; and Supplemental Figure 1A available at http://ajp.amjpathol.org). There was some faint cloudy staining around the major interlobular bile ducts (Figure 3A, and Supplemental Figure 1A available at http://ajp.amjpathol.org). Thy-1 antibody decorated more intensely and sharply the cross sections of peripheral nerves (Figure 3A, and Supplemental Figure 1A available at http://ajp.amjpathol.org). These nerves were also positive for synaptophysin (data not shown). Scattered undefined cells inside the portal areas also expressed Thy-1 (Figure 3, C and E). Desmin antibody reacted with nonparenchymal cells in the liver lobule in addition to the muscular wall of the blood vessels and scattered single cells in the periportal connective tissue (Figure 3, C and D). Conversely, no SMA-positive cells were seen inside the liver lobule; only the vessel walls were stained (Figure 3, D and E). Glial fibrillary acidic protein (GFAP), an established marker of hepatic stellate cells, decorated scattered single cells in the liver lobules. There was no overlap between the GFAP and Thy-1 reaction (Figure 3B). In rat liver treated according to the AAF/PHx protocol, oval cells formed ductules invading the liver lobules during the first 7 to 10 days. These ductules are surrounded by continuous basement membrane, and there are numerous stellate cells/myofibroblasts around them.15Paku S Schnur J Nagy P Thorgeirsson SS Origin and structural evolution of the early proliferating oval cells in rat liver.Am J Pathol. 2001; 158: 1313-1323Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar The number of transit-amplifying oval cells depends on the dose of AAF as demonstrated by the levels of AFP transcripts—the most widely used marker for rat oval cells (Figure 1A). A similar expression pattern was found in whole liver for Thy-1, confirming that Thy-1 expression is induced during oval cell-mediated liver regeneration (Figure 1A). However, when isolated oval cells from AAF/PHx-treated animals were examined, no expression of Thy-1 was detectable despite increased levels of AFP transcripts (Figure 1B). qRT-PCR also failed to detect Thy-1 (and SMA) expression in RNA isolated from microdissected oval cells, while AFP RNA was present. However, Thy-1 and SMA expression could be demonstrated by qRT-PCR from whole liver sections. Therefore, we performed a thorough immunohistochemical analysis of the Thy-1 expression. The staining pattern with the different antibodies was identical in all studied time points. The laminin-containing basement membrane surrounded the CK-19-positive oval cell ductules. The Thy-1 reaction was observed consistently outside the basement membrane (Figure 3, F and G; and Supplemental Figure 1, B and C, available at http://ajp.amjpathol.org). The antibody sometimes labeled round, cellular body-like elements, but frequently only stripes or cell processes were positive. Thy-1 immunoreactions were easily abolished by detergent pretreatment. If sections were pretreated for 5 minutes in 0.05% Triton X-100, the staining was faint whereas pretreatment for 10 minutes resulted in complete disappearance of the reaction (data not shown). Thy-1 antibody also decorated cellular elements and long processes outside the basement membrane in human livers with extensive ductular reactions because of fulminant liver failure (Figure 3H). The pattern of Thy-1 reaction was reminiscent of stellate cell/myofibroblast architecture, which also could be found outside the basement membrane. Therefore, in the rat liver we performed co-staining of Thy-1 and SMA or desmin, the two most widely used stellate cell/myofibroblast markers. SMA-positive cells appeared very early in the experiment at the limiting plate and spread along the ductules formed by oval cells into the parenchyma. The desmin antibody reacted with scattered nonparenchymal cells throughout the liver lobule from the beginning of the experiment, but they became more frequent in the zone of the oval cells. In the co-staining experiments, Thy-1 showed frequent co-localization with SMA (Figure 4, A–C). Of the Thy-1-positive areas 80.3 ± 9.6% stained with SMA, but only 58 ± 9.3% of the SMA-positive field was decorated by Thy-1. Thy-1 positivity hardly overlapped with desmin; the value of co-localization index was 6.8 ± 2.3% (Figure 4D and Supplemental Movie 1 available at http://ajp.amjpathol.org). This was surprising because SMA and desmin have been used to identify stellate cells/myofibroblasts. The co-localization index of these two markers was also negligible (7.16 ± 1.2%) (Figure 4E). To investigate Thy-1 co-localization with other marker antigens, we performed further double-staining experiments. Lyve-1, a new marker for the endothelial cells of lymphatic vessels and hepatic sinusoids,16Banerji S Ni J Wang SX Clasper S Su J Tammi R Jones M Jackson DG LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.J Cell Biol. 1999; 144: 789-801Crossref PubMed Scopus (1320) Google Scholar, 17Mouta Carreira C Nasser SM di Tomaso E Padera TP Boucher Y Tomarev SI Jain RK Lyve-1 is not restricted to the lymph vessels: expression in normal liver blood sinusoids and down-regulation in human liver cancer and cirrhosis.Cancer Res. 2001; 61: 8079-8084PubMed Google Scholar did not show any co-staining with Thy-1 in the neighborhood of the oval cells (Figure 4F), a result that was similar to OX 62 and rMPh markers of hepatic dendritic18Brenan M Puklavec M The MRC OX-62 antigen: a useful marker in the purification of rat veiled cells with the biochemical properties of an integrin.J Exp Med. 1992; 175: 1457-1465Crossref PubMed Scopus (213) Google Scholar and Kupffer cells (data not shown). In addition, CD45, a general leukocyte marker, did not stain the Thy-1-positive structures (Figure 4G). Immunoelectron microscopic examination of Thy-1 expression also revealed long cell processes running clearly outside the basement membrane. Because of immunoelectronmicroscopic processing of the samples, the ultrastructure of labeled cells could not be examined in detail. However, our morphological evaluation suggested that the marked cells displayed features of stellate cells/myofibroblasts (Figure 5, A and B). Thy-1 also decorated the myofibroblasts in two liver fibrosis models (bile duct ligation-induced cholangiofibrosis and CCl4/phenobarbital-induced cirrhosis), which were not characterized by oval cell proliferation (Figure 6). We have investigated Thy-1 expression in rat livers regenerating by the recruitment of oval/progenitor cells. The oval cells were not labeled by the Thy-1 antibody, but we observed a strong periductal reaction outside the basement membrane. There was a partial overlap between the Thy-1 and SMA staining, but no co-staining of Thy-1 and desmin could be observed. Furthermore, Thy-1 also marked myofibroblasts in two liver fibrosis models without oval cell reactions. Thy-1 is a highly conserved protein anchored by a phosphatidylinositol to the cell membrane. Its exact function is unknown, but it has been proposed to be involved in cell recognition, adhesion, and lymphocyte activation.1Petersen BE Goff JP Greenberger JS Michalopoulos GK Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat.Hepatology. 1998; 27: 433-445Crossref PubMed Scopus (337) Google Scholar It is expressed in a wide variety of different tissues.1Petersen BE Goff JP Greenberger JS Michalopoulos GK Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat.Hepatology. 1998; 27: 433-445Crossref PubMed Scopus (337) Google Scholar, 19D'Arena G Musto P Cascavilla N Carotenuto M Thy-1 (CDw90) and c-kit receptor (CD117) expression on CD34+ hematopoietic progenitor cells: a five dimensional flow cytometric study.Haematologica. 1998; 83: 587-592PubMed Google Scholar, 20Boiret N Rapatel C Boisgard S Charrier S Tchirkov A Bresson C Camilleri L Berger J Guillouard L Guerin JJ Pigeon P Chassagne J Berger MG CD34+CDw90(Thy-1)+ subset colocated with mesenchymal progenitors in human normal bone marrow hematon units is enriched in colony-forming unit megakaryocytes and long-term culture-initiating cells.Exp Hematol. 2003; 31: 1275-1283Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 21Timper K Seboek D Eberhardt M Linscheid P Christ-Crain M Keller U Muller B Zulewski H Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells.Biochem Biophys Res Commun. 2006; 341: 1135-1140Crossref PubMed Scopus (393) Google Scholar, 22Saalbach A Hildebrandt G Haustein UF Anderegg U The Thy-1/Thy-1 ligand interaction is involved in binding of melanoma cells to activated Thy-1-positive microvascular endothelial cells.Microvasc Res. 2002; 64: 86-93Crossref PubMed Scopus (44) Google Scholar, 23Koumas L Smith TJ Feldon S Blumberg N Phipps RP Thy-1 expression in human fibroblast subsets defines myofibroblastic or lipofibroblastic phenotypes.Am J Pathol. 2003; 163: 1291-1300Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar Its expression has been extensively studied in the liver. Petersen and colleagues1Petersen BE Goff JP Greenberger JS Michalopoulos GK Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat.Hepatology. 1998; 27: 433-445Crossref PubMed Scopus (337) Google Scholar reported that hepatic oval cells expressed the hematopoietic stem cell marker Thy-1 in the rat. This observation led to further experiments suggesting that bone marrow cells can be the precursors of oval cells/hepatocytes. Hepatic progenitor cells in human fetal liver also have been reported to be Thy-1-positive.7Masson NM Currie IS Terrace JD Garden OJ Parks RW Ross JA Hepatic progenitor cells in human fetal liver express the oval cell marker Thy-1.Am J Physiol Gastrointest Liver Physiol. 2006; 291: G45-G54Crossref PubMed Scopus (81) Google Scholar However, Hoppo and colleagues9Hoppo T Fujii H Hirose T Yasuchika K Azuma H Baba S Naito M Machimoto T Ikai I Thy1-positive mesenchymal cells promote the maturation of CD49f-positive hepatic progenitor cells in the mouse fetal liver.Hepatology. 2004; 39: 1362-1370Crossref PubMed Scopus (64) Google Scholar found in mouse that Thy-1-positive mesenchymal cells promoted the maturation of Thy-1-negative hepatic progenitor cells. A subpopulation of the Thy-1-positive cells also expressed SMA. Our results in the rat are similar to this latter group's observation. In our case the CK-19-positive, laminin-surrounded oval/progenitor cells were not decorated by the Thy-1 antibody. CK-19 is an established marker of oval cells. It is also generally accepted that the oval cells are surrounded by continuous basement membrane, which can be visualized by laminin immunohistochemistry. The co-staining of these three antigens on the same section combined by confocal microscopic analysis is a very reliable morphological examination. Thy-1-positive cells were also outside the proliferating ductules in human liver, confirming the observation of Crosby and colleagues.24Crosby HA Nijjar SS de Goyet J de V Kelly DA Strain AJ Progenitor cells of the biliary epithelial cell lineage.Semin Cell Dev Biol. 2002; 13: 397-403Crossref PubMed Scopus (50) Google Scholar Furthermore, immunoelectronmicroscopy confirmed that the Thy-1-positive cells are outside the basement membrane. On traditional histological sections, it is very difficult to distinguish conclusively between the oval and the closely associated stellate cells/myofibroblasts. Serial sections stained by an oval cell marker, which was used by Petersen and colleagues,1Petersen BE Goff JP Greenberger JS Michalopoulos GK Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat.Hepatology. 1998; 27: 433-445Crossref PubMed Scopus (337) Google Scholar do not provide much help. Individual cells cannot be analyzed on serial sections, and the histological arrangement of the two cell populations is similar (spreading outward from the periportal region). Our Northern hybridization and qRT-PCR analysis also strongly support the morphological observations. The Thy-1 molecule is bound weakly to the cell membrane.25Heffer-Lauc M Lauc G Nimrichter L Fromholt SE Schnaar RL Membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation.Biochim Biophys Acta. 2005; 1686: 200-208Crossref PubMed Scopus (62) Google Scholar, 26Heffer-Lauc M Viljetic B Vajn K Schnaar RL Lauc G Effects of detergents on the redistribution of gangliosides and GPI-anchored proteins in brain tissue sections.J Histochem Cytochem. 2007; 55: 805-812Crossref PubMed Scopus (32) Google Scholar Triton X-100 pretreatment in our study also deleted the Thy-1 signal from the sections. Soluble Thy-l has been also described in the serum.27Saalbach A Wetzig T Haustein UF Anderegg U Detection of human soluble Thy-1 in serum by ELISA: fibroblasts and activated endothelial cells are a possible source of soluble Thy-1 in serum.Cell Tissue Res. 1999; 298: 307-315Crossref PubMed Scopus (70) Google Scholar Enzymatic digestion during the cell isolation procedures may cause detachment of the Thy-1 molecule, which might associate later to other cells causing misleading results in cell suspension. To identify the Thy-1-expressing cells, we co-stained Thy-1 with several other marker antibodies. The negative results with OX-62, rMPh, and Lyve-1 excluded hepatic dendritic, Kupffer, and sinusoidal endothelial cells as the sources of Thy-1 expression. The position and shape of the Thy-1-positive cells refers strongly to the so-called stellate cells/myofibroblasts, which are well known to have close spatial relationship with the oval cells in all oval cell proliferation models. The partial co-staining with SMA supports this option, as well as the Thy-1 positivity of myofibroblasts in two hepatic fibrosis models. Thy-1-positive myofibroblasts were described in other tissues, and the ratio of the Thy-1+/− populations was a function of their activation stage.28Sanders YY Kumbla P Enhanced myofibroblastic differentiation and survival in Thy-1(−) lung fibroblasts.Am J Respir Cell Mol Biol. 2007; 36: 226-235Crossref PubMed Scopus (116) Google Scholar, 29Hudon-David F Bouzeghrane F Couture P Thibault G Thy-1 expression by cardiac fibroblasts: lack of association with myofibroblast contractile markers.J Mol Cell Cardiol. 2007; 42: 991-1000Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 30Rege TA Hagood JS Thy-1, a versatile modulator of signaling affecting cellular adhesion, proliferation, survival, and cytokine/growth factor responses.Biochim Biophys Acta. 2006; 1763: 991-999Crossref PubMed Scopus (142) Google Scholar, 31Hagood JS Prabhakaran P Kumbla P Salazar L MacEwen MW Barker TH Ortiz LA Schoeb T Siegal GP Alexander CB Pardo A Selman M Loss of fibroblast Thy-1 expression correlates with lung fibrogenesis.Am J Pathol. 2005; 167: 365-379Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar Surprisingly, co-staining of Thy-1 with desmin, another marker of stellate cells, could not be demonstrated. Desmin and SMA are alternatively used markers for stellate cells.32Ramadori G Saile B Mesenchymal cells in the liver—one cell type or two?.Liver. 2002; 22: 283-294Crossref PubMed Scopus (99) Google Scholar These antibodies stain comparably located and shaped cell populations, but according to our result, the two reactions do not overlap. The origin and phenotype of hepatic stellate cells/myofibroblasts is one of the most controversial issues of liver pathobiology. It is not known if there are different differentiation/activation stages of the same cell population, or as Ramadori and Saile32Ramadori G Saile B Mesenchymal cells in the liver—one cell type or two?.Liver. 2002; 22: 283-294Crossref PubMed Scopus (99) Google Scholar propose, there are two (or more) cell types with partially overlapping phenotype. Our observations on the normal liver and during the progression of oval cell proliferation in the rat are in support of the view of Ramadori and Saile.32Ramadori G Saile B Mesenchymal cells in the liver—one cell type or two?.Liver. 2002; 22: 283-294Crossref PubMed Scopus (99) Google Scholar Scattered desmin/GFAP-positive cells were observed in the parenchyma of the normal liver, which might correspond to the (classical, perisinusoidal, vitamin A-storing) stellate cells. SMA-decorated cells were confined to the vessel walls in the normal liver, but they appeared in the periportal region very early after treatment with AAF alone (data not shown) and later spread along the oval cell ductules. This would imply that the SMA-positive cells would be myofibroblasts, derived from the periportal fibroblasts, as proposed by Ramadori and Saile32Ramadori G Saile B Mesenchymal cells in the liver—one cell type or two?.Liver. 2002; 22: 283-294Crossref PubMed Scopus (99) Google Scholar and Beaussier and colleagues.33Beaussier M Wendum D Schiffer E Dumont S Rey C Lienhart A Housset C Prominent contribution of portal mesenchymal cells to liver fibrosis in ischemic and obstructive cholestatic injuries.Lab Invest. 2007; 87: 292-303Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar As far as we know, co-staining for SMA and desmin has not been published in oval cell proliferation experiments. Considering the above data, the Thy-1-positive cells in the zone of the oval cells show the closest association with the myofibroblasts. The increased expression of Thy-1 in two liver fibrosis models also supports the myofibroblastic origin of this marker molecule. The appearance of Thy-1-positive cells in the liver parenchyma can be explained if some of the myofibroblasts acquire the Thy-1 expression during the invasion of the liver lobule. Alternatively, it has been found that the mesenchymal stem cells in the bone marrow are Thy-1-positive and that this cell compartment can contribute to wound-healing processes34Tomasek JJ Gabbiani G Hinz B Chaponnier C Brown RA Myofibroblasts and mechano-regulation of connective tissue remodelling.Nat Rev Mol Cell Biol. 2002; 3: 349-363Crossref PubMed Scopus (3228) Google Scholar including the fibrogenesis of the liver.35Forbes SJ Russo FP Rey V Burra P Rugge M Wright NA Alison MR A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis.Gastroenterology. 2004; 126: 955-963Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar It cannot be excluded that the Thy-1 antibody recognizes bone marrow-derived mesenchymal cells, which may participate in oval cell-mediated liver regeneration. At present, we cannot distinguish between these two possibilities, but transplantation experiments are under way to study the presence of bone marrow-originated cells among the stellate cells/myofibroblasts. Recently, Kisseleva and colleagues36Kisseleva T Uchinami H Feirt N Quintana-Bustamante O Segovia JC Schwabe RF Brenner DA Bone marrow-derived fibrocytes participate in pathogenesis of liver fibrosis.J Hepatol. 2006; 45: 429-438Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar have described a unique CD45+ fibrocyte population in the liver. However, in accordance with our results, Kamo and colleagues37Kamo N Yasuchika K Fujii H Hoppo T Machimoto T Ishii T Fujita N Tsuruo T Yamashita JK Kubo H Ikai I Two populations of Thy1-positive mesenchymal cells regulate the in vitro maturation of hepatic progenitor cells.Am J Physiol Gastrointest Liver Physiol. 2007; 292: G526-G534Crossref PubMed Scopus (25) Google Scholar could not demonstrate co-expression of Thy-1 and CD45. In conclusion, we did not find Thy-1 expression in the hepatic oval/progenitor cell population in stem cell-mediated rat liver regeneration or in human ductular reactions. Instead, Thy-1 was localized to a subpopulation of stellate cells/myofibroblasts. Therefore, the use of Thy-1 as a cell surface marker for isolation of oval/progenitor cells from the liver is not recommended. The exact origin and function of Thy-1-expressing cells remains to be studied. Our results are in complete agreement with the recently published study of Dudas and colleagues.38Dudas J Mansuroglu T Batusic D Saile B Ramadori G Thy-1 is an in vivo and in vitro marker of liver myofibroblasts.Cell Tissue Res. 2007; 329: 503-514Crossref PubMed Scopus (61) Google Scholar We thank Sándor Spisák for helping in microdissection. 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